The invention is in the field of medicinal engineering and concerns a method according to the generic part of the first independent claim, i.e. a method for producing cartilage tissue and implants for the repair of enchondral and osteochondral defects. Furthermore, the invention concerns an arrangement for carrying out the method and implants produced according to the method.
Cartilage tissue substantially consists of chondrocytes and extracellular matrix. The extracellular matrix mainly consists of collagen type II and proteoglycanes the components of which are exuded into the intercellular space where they are assembled to form macro molecules. The chondrocytes make up about 5% of the volume of the cartilage tissue of a grown-up individual.
Articular cartilage coating the ends of flexibly joined bones takes over the function of the load distribution in the loaded joint. For this function the cartilage tissue is capable to take up water and to release it again under pressure. Furthermore, the cartilage surfaces serve as sliding surfaces in the joints.
Cartilage is not vascularized and therefore its ability to regenerate is very poor, in particular in grown-up individuals and if the piece of cartilage to be regenerated exceeds but a small volume. However, articular cartilage often shows degenerations due to wear or age or injuries due to accidents with a far larger volume than might be naturally regenerated. This kind of defect of the cartilage layer makes movement and strain of the affected joint painful and can lead to further complications such as e.g. inflammation caused by synovial liquid which comes into contact with the bone tissue due to the defect in the cartilage layer covering the bone.
For these reasons efforts have been made for quite some time to replace or repair missing or damaged cartilage, especially articular cartilage by corresponding surgery.
It is known to repair defects concerning articular cartilage or articular cartilage and the bone tissue beneath it by milling the defect location to form a bore of an as precise geometry as possible, by extracting a column of cartilage and bone of the same geometry from a less strained location of e.g. the same joint by means of boring or punching and by inserting this column into the bore. In the same manner, larger defects with several bores are repaired (mosaic plasty). These methods are successful but the actual problem is substantially shifted from a strained part of a joint to a less strained part of the joint and therefore, is not really solved.
It is also suggested, e.g. in the publication U.S. Pat. No. 3,703,575 (Thiele), to repair defects of cartilage with purely artificial implants (e.g. gels containing proteins and polysaccharides). It shows, however, that only restricted success can such be achieved and therefore in recent development solutions to the problem have been thought in various directions, in particular based on vital autologous or homologue cells. Vital chondrocytes or cells able to take over a chondrocyte function are e.g. cultivated in vitro and then implanted; or vital chondrocytes are introduced in artificial implants; or vital cartilage tissue is cultivated at least partly in vitro and is then implanted. This means that in these recent developments the aim is to produce vital cartilage in vitro and to implant such cartilage or to populate a defect site with cartilage forming cells which cells are then to build tissue at least similar to cartilage.
Examples of such methods are described in the following publications:
According to the method described in U.S. Pat. No. 4,846,835 (Grande), chondrocytes taken from the patient are multiplied in a mono-layer culture and, for further reproduction, are then introduced into a three-dimensional collagen matrix in form of a gel or a sponge in which matrix they settle and become immobile. After ca. three weeks of cell reproduction, the defect cartilage location is filled with the material consisting of the collagen matrix and the cells. In order to hold the implant in the defect location, a piece of periosteum is sutured over it. The cartilage regeneration in the region of this kind of transplant is considerably better than without the transplant.
According to the method described in U.S. Pat. No. 5,053,050 (Itay), chondrocytes or cells able to take over a chondrocyte function are introduced into a bio-compatible, resorbable matrix (32xc3x97106 to 120xc3x97106 cells per cm3) in which matrix the cells are immobilized. This matrix is implanted, whereby a cartilage-like tissue forms in vivo. The chondrocytes used for the implant are previously cultivated, first in a mono-layer culture and then suspended, whereby they assemble to form aggregates of 30 to 60 cells.
According to the method described in U.S. Pat. No. 4,963,489 (Naughton), again a three-dimensional, artificial matrix is used as carrier material for the implant. This matrix is used for the cell culture preceding the implantation and is covered with a layer of connective tissue for better adhesion and better supply of the cells to be cultivated. After in vitro cell reproduction on the three-dimensional matrix, the matrix is implanted. The implanted cells form the cartilage tissue in vivo.
According to the method described in PCT-WO90/12603 (Vacanti et al.), again a three-dimensional matrix is used which matrix consists of degradable polymer fiber materials and on which matrix the cells settle. The cells cultivated on the matrix or in monolayer cell cultures and then introduced into the matrix are implanted adhering to the matrix and therefore, in an immobilized state. The matrix is degraded in vivo and is gradually replaced by extracellular matrix built by the cells.
According to the method described in U.S. Pat. No. 5,326,357 (Kandel), chondrocytes are applied to a layer of filter material (MILLICELL(copyright)-CM having a pore size of 0.4 xcexcm) in a mono-layer with a cell density of 1.5xc3x97106 cells per cm2. In vitro culturing of the monolayer produces a thin cartilage layer in two to four weeks which, in its structure obviously corresponds to the natural articular cartilage and can be implanted as such.
It is also known that cartilage can be cultivated in so called high density cell cultures. Cells are applied to a carrier and are cultured in a higher density than used for mono-layer culturing. The culture medium is added only one to two hours after bringing the cells onto the carrier. After one to three culture days, the cell layer on the carrier contracts and so-called microspheres with diameters in the range of 1 mm form. On further culturing, a cartilage-like tissue forms inside these microspheres while fibrous cartilage (perichondrium) forms on their surface. For implants, this kind of inhomogeneous tissue is not suitable.
Sittinger et al. (Biomaterials Vol. 17, No. 10, May 1996, Guilford GB) suggest to introduce vital cells into a tree-dimensional matrix for growing cartilage in vitro and to then enclose the loaded matrix into a semi-permeable membrane. During the cartilage growth, this membrane is to prevent the culture medium to wash away compounds produced by the cells and being used for constructing the extracellular matrix. Implantation of cell cultures enclosed in this kind of membranes is also known for preventing immune reactions.
All methods named above attempt to produce cartilage at least partly in vitro, i.e. to produce cartilage using vital natural cells under artificial conditions. The problem encountered in these attempts is the fact that chondrocytes in these in vitro conditions have the tendency to de-differentiate into fibroblasts relatively rapidly, or the fact that it is possible to differentiate fibroblasts to a chondrocyte function under very specific culture conditions only. By the dedifferentiation the chondrocytes among other things loose the ability to produce type II collagen which is one of the most important compounds of cartilage tissue.
According to the methods mentioned above, the problem of de-differentiation of chondrocytes is solved by immobilizing the chondrocytes in correspondingly dense cultures in a monolayer or in a three-dimensional matrix. It shows that in this manner chondrocytes reproduce themselves without substantial de-differentiation and form an extracellular matrix which is at least similar to the extracellular matrix of natural cartilage. The three-dimensional matrix is mostly not only used for immobilizing the cells but also for mechanical stability after implantation which is needed because none of the cartilage tissues produced in the named manner has a stability which could withstand even a greatly reduced strain.
The object of the invention is to create a device with which suitable cartilage tissue or implants which at least partly consist of such cartilage tissue can be produced in vitro, the cartilage or implant serving for implantation, especially implantation in articular cartilage defects. For achieving this object it is necessary to create an in vitro environment, in particular a three-dimensional such environment, in which environment chondrocytes or other, cells capable of a chondrocyte function do not de-differentiate over a longer culture period and perform their function actively or in which environment cells differentiate to become active chondrocytes respectively. By solving the problem of this environment, the main part of the object is achieved as not de-differentiating, vital chondrocytes according to their natural function produce the extracellular matrix characteristic for cartilage and together with this form the cartilage tissue to be created.
The implants produced according to the invention consist at least partly of cartilage tissue produced in vitro and are especially suited for the repair of enchondral or osteochondral joint defects. They are to be producible for any possible depth of such a defect and a defect repaired with the inventive implant is to be able to carry a normal load as soon as possible after implantation, i.e. a load created either by pressing or by shearing forces.
This object is achieved by the method as defined in the claims.
The inventive. method is based on the finding that chondrocytes can build satisfactory cartilage tissue if it is made possible that a sufficiently high concentration of compounds produced by the cells and segregated into extracellular spaces is achieved in a short initial phase and is maintained during the whole culture period. Under these conditions the differentiated function of the chondrocytes is fully maintained (they do not de-differentiate into fibroblasts) and/or it is possible to differentiate corresponding cells, especially mesenchymal stem cells or other mesenchymal cells or even fibroblasts to a corresponding function.
The first condition is fulfilled by creating a cell community in which there is, at least at the beginning of the culture period, a density of cells such that the cells are capable to produce the amount of compounds necessary for the mentioned concentrations in the spaces between the cells. The second condition is fulfilled by accommodating the cell community in a restricted cell space in which washing out of the named compounds from the extracellular spaces is prevented.
The named compounds are especially autocrine factors and substances serving as components for building the extracellular structure. These components are especially aggrecanes, link-proteins and hyalorunates for building proteo-glycane-aggregates and preliminary stages of collagens to eventually form collagen-fibrils of type II.
According to the inventive method, the cells are not immobilized for the in vitro culture but have space at their disposition, space without a three-dimensional, artificial matrix in which space the two conditions mentioned above are fulfilled, in which space it is however largely left to the cells how they are to settle relative to each other. It shows that in this kind of free cell space, cells fully practice their chondrocyte-function and a cartilage tissue having sufficient stability for implantation and being able to carry at least part of the normal load after implantation can be cultured.
According to the inventive method, cells which are capable of a chondrocyte-function are introduced into an empty cell space, i.e. into a space containing culture medium only, such that there is a cell density of ca. 5xc3x97107 to 109 cells per cm3 in the cell space. This density amounts to a space occupation of ca. 5% to 100% at an approximate cell volume of 103 xcexcm.
The cell space has at least partly permeable walls and is introduced into a space filled with culture medium for the length of the culture period which medium is periodically renewed in known manner. During the culture period, the cell space is arranged to be stationary in the culture medium or it is moved in it (relative movement between cell space and culture medium surrounding the cell space).
The permeability of the permeable parts of the cell space wall and the relative movement are to be matched to the relative dimension of the cell space (depending on the cartilage to be produced) such that the condition of the washing-out-prevention is fulfilled.
For all cases, semi-permeable wall regions (semi-permeable membranes) with a permeability of 10,000 to 100,000 Dalton (10 to 100 kDa) are suitable, especially for agitated cultures and for cell spaces of large dimensions (three-dimensional forms). The named autocrine factors and components for building the macromolecules of the extracellular cartilage matrix have molecular weights which are such that they cannot pass through a membrane with the named permeability.
It shows that for stationary cultures and cell spaces with at least one small dimension (thin layers) open-pore walls with considerably larger pores (up to the region of 10 to 20 xcexcm) which cannot be effective as semi-permeable walls but merely as convection barriers are sufficient and that possibly the cell space can even be open on one side.
Especially in cell spaces not being moved and containing cells at a density in the lower region of the given density range, the cells settle in the direction of gravity and form cartilage tissues in the form of layers. For producing more three-dimensional cartilage forms, agitated cell spaces prove to be advantageous.
For specific cultures and especially for specific forms and sizes of cell spaces the optimal arrangement (with or without movement of the cell space in the culture medium) and the optimal condition of the wall of the cell space or the permeable parts of this wall respectively must be determined by experiment.
The cell space has substantially three functions:
The cell space keeps the community of the (not immobilized) cells together at a sufficient density such that they do not loose their specific functionality;
the cell space restricts the growth of cartilage such that by choice of the form of the cell space the form of the cartilage being formed is controlled;
the cell space wall allows the supply of the cells with culture medium but prevents the washing out of the substances produced by the cells and necessary for the growth of cartilage.
For producing implants which only partly consist of cartilage tissue, a part of the permeable wall of the cell space can additionally have the function of an implant part as will be described in more detail further below.
The cells to be brought into the cell space are chondrocytes, mesenchymal stem cells or other mesenchymal cells. These cell types are isolated in known manner from cartilage tissue, from bone or bone marrow or from connective tissue or fatty tissue. Fibroblasts are also suited, e.g. if factors are added to the culture medium or the cell space which factors effect differentiation of the fibroblasts to chondrocytes or if the cells are treated with this kind of factor before they are brought into the cell space. The cells can also be multiplied in vitro before being brought into the cell space.
It is not necessary to isolate specific cell types from donor tissue, i.e. mixtures of different cells as usually contained in such tissues can be brought into the cell space as such. It also shows that a complete separation of the cells from the intracellular matrix of the donor tissue is not necessary and thus possibly tissue particles or mixtures of isolated cells and tissue particles can be brought into the cell space instead of cells only. However, care has to be taken that the necessary cell density is achieved in the cell space possibly by partly separating the. cells from their extracellular matrix, e.g. by means of a short enzymatic digestion.
It shows that with the known culture media such as e.g. HAM-F12 to which 5 to 15% serum is advantageously added, good results can be achieved. Furthermore, known growth factors and other components of culture media which support the reproduction of the cells and the forming of the cartilage matrix can be added to the culture medium.
It shows that in the culture conditions created according to the inventive method the chondrocytes remain active and do not de-differentiate such that the space is filled with cartilage tissue in a culture period in the range of ca. three weeks. The cartilage tissue being formed can, as soon as it has a sufficient mechanical strength, be removed from the cell space and e.g. be further cultivated floating freely in the culture medium or it can remain in the cell space up to directly before being used.
Cartilage tissue produced according to the inventive method is used as implant as implant part or when containing autologous cells as cell-auto-transplant or it can be used for scientific in vitro purposes.